Many important cellular functions are performed by large complexes which operate as macromolecular machines. Complexes also play primarily structural roles as biomaterials in many tissues, including skin and muscle. We aim to elucidate the structures, assembly properties, and interactions of complexes of both kinds, with close attention to the functional connotations. Over the past yeasr, we pursued three subprojects. (1) Energy-dependent Proteases. Protein quality control is essential for eliminating aberrant proteins that would otherwise pollute the cell, for example by amyloid formation. This activity is largely carried out by energy-dependent machines which generically consist of two subcomplexes - a peptidase and a chaperone-like ATPase. Our studies focus on the Clp proteases of E. coli which offer a tractable model system. Earlier we showed that peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. The ATPases stack axially on one or both faces of ClpP. We went on to study the interaction of ClpAP and ClpXP with substrate proteins which we found to bind to distal sites on the ATPase before being translocated axially into the digestion chamber inside ClpP. New Results. (i) We have focused on reconciling our cryo-EM reconstruction of the fully assembled ClpA hexamer (ATPgS state) at 1.2 nm resolution with the published crystal structure of the ClpA monomer (ADP state). There is good agreement in the hexameric ring of D1 ATPase domains, which is the most static part of the structure. The D2 ring, which has higher ATPase activity, shows discrepancies in the interior, suggesting local mobility, and the 17 kDa N-terminal domains exhibit large (> 3.5nm) random motions around median positions that are far removed from the crystal conformation. We infer that the latter domains are highly mobile, and posit that this mobility is exploited in the interaction with substrates. (ii) We have also studied the binding of the 20S proteasome (its peptidase) with the PA200 activity. We find that a 200-kDA monomer of PA200 binds to one or both ends of the 20S core and in so doing, opens the exial channel to facilitate the passage of substrates or digested peptides. Bioinformatic analysis of PA200 detected a novel class of HEAT repeats, suggesting that the molecular structure is a solenoid of these alpha-helical motifs. (2) Amyloid filament formation by the yeast prion protein, Ure2p. Amyloid is fibrous aggregates of protein(s) in protease-resistant, beta-sheet-rich, non-native conformations. Amyloid accumulates in a number of disease situations including rheumatoid arthritis. Prions (infectious proteins) are transmissible amyloids implicated in certain neuropathies, notably the spongiform encephalopathies. To investigate the structure of amyloids and the mechanisms that underlie their formation, we study yeast prions. Unlike mammalian prions, their phenotypes are expressed via metabolic functions rather than cytopathic effects. This greatly simplifies and accelerates their study. We focus on Ure2p, a protein involved in nitrogen metabolism. Its prion phenotype presents as an inability to grow on poor nitrogen sources. In earlier work, we demonstrated filament formation by Ure2p in vitro and the presence of filaments in prion-infected cells. New Results: Over the past year, we further substantiated our ?amyloid backbone" model, according to which, the N-terminal "prion" domains polymerize to form an amyloid backbone to Ue2p filaments that is surrounded by the natively folded C-terminal domains, whereas in soluble Ure2p, the N-domains are unfolded. This model successfully predicted that fusions of the prion domain with exogenous proteins should also form filaments. We have now analyzed several Ure2p constructs by differential scanning calorimetry, with complementary EM, and found that the C-terminal domain is indeed conformationally indistinguishable in soluble and filamentous Ure2p. The data also support the N-terminus being unfolded in soluble Ure2p and so tightly folded in filaments as to resist thermal denaturation up to 105 degrees C. We also used electron diffraction and X-ray fiber diffraction to demonstrate a reflection at a spacing of 0.47nm that is typical for cross-beta structures, and we developed a novel model for the amyloid backbone. This model, which we all a "beta super-pleated structure" envisages an array of parallel beta-sheets with each subunit adopting a "beta-serpentine" fold that accounts for all current data on Ure2p filaments and is adaptable to several other amyloids. We have also used electron tomography of thin sections to investigate aggregates of Ure2p filaments in infected yeast cells and as assembled in vitro. These observations find the respective filaments to be essentially indistinguishable, thereby supporting the view that our in vitro experiments correctly mimic prionogenesis in vivo. (3) Structural Basis of Actin-Based Motor Activity. Acanthamoeba myosin IC (AMIC) is a monomeric, single-headed, myosin comprising one heavy chain (129 kDa) and one light chain (17 kDa) that associates with intracellular membranes. In previous work, we investigated the effects of heavy chain phosphorylation in activating its ATPase. In the past year, we went on to determine the organization of subdomains in the AMIC tail by cryo-EM and image reconstruction. The heavy chain has head, neck (light chain-binding), and tail domains. The tail consists of four subdomains: a basic region (23 kDa) and two Gly/Pro/Ala-rich regions - GPA1 (6 kDa) and GPA2 (15 kDa), flanking an SH3 region (6 kDa). New Results. To determine their spatial arrangement, we compared actin filaments decorated with wild-type AMIC and with tail-truncated mutants of various lengths. The basic region forms an oval-shaped feature, ~ 4nm long, that diverges obliquely from the head, extending azimuthally around the actin filament and towards its barbed end. GPA2 and GPA1 are located together on the inner (actin-proximal) side of the tail, close enough to act in concert in binding the same or another actin filament. The outer face of the basic region is strategically exposed for membrane or vesicle binding.